Posttranslational modifications play important roles in many signal transduction pathways, and we have yet to realize the full extent to which these modifications impact on cell function. Recent studies have highlighted a key role for a distinctive modification, prolyl hydroxylation, in the hypoxic response. In this application, we propose to develop and employ novel capture reagents for this posttranslational modification. The master transcriptional regulator of the hypoxic response in metazoans is Hypoxia Inducible Factor (HIF), which consists of an and a subunit. The key event in oxygen-regulated HIF turnover is posttranslational modification of HIF- by site-specific prolyl hydroxylation, which allows recognition by the von Hippel Lindau (VHL) tumor suppressor protein, a component of an E3 ubiquitin ligase complex that selectively targets hydroxylated HIF- for degradation. This, in turn, raises the larger question of whether prolyl hydroxylation may play a broader role in the hypoxic response. A major limitation to addressing this question is the lack of suitable reagents that can specifically recognize prolyl hydroxylated proteins. We propose developing novel hydroxylprolyl capture reagents using VHL as a scaffold for introducing site- directed mutations. These mutations will preserve the hydroxylprolyl binding pocket of VHL with the intention of extending reactivity to other hydroxylprolyl-containing proteins. The VHL-based hydroxylprolyl capture reagents will be employed to immunoprecipitate proteins, and their identity and sites of prolyl hydroxylation will be obtained by mass spectrometry. Studies employing cell culture and mouse tissue extracts will be employed to characterize the significance of these prolyl hydroxylation events. Platforms for assessing changes in prolyl hydroxylation in a high throughput manner will be developed. Our collaborator, Dr. Stephen Master, will focus on these high throughput studies and the mass spectrometry analyses. These studies will challenge the current paradigm that the role of prolyl hydroxylation in hypoxic responses is limited to HIF. Moreover, they will provide the basis for assessing dynamic changes in the hydroxylproteome, which is of considerable significance given the role of hypoxia in diseases that include myocardial infarction, cerebrovascular disease, and cancer.